Platelet-derived growth factor (PDGF) was identified more than three decades ago as a serum growth factor for fibroblasts, smooth muscle cells and glia cells. Its role in physiology and medicine is extensively described in a recent review (Andrae, J., Gallini, R. and Betsholtz, C., Genes Dev., 22, 1276-1312, 2008). Human PDGF was originally identified as a disulfide-linked dimer of two different polypeptide chains, A (PDGF-A; human PDGF-A has the UniProtKB/Swiss-Prot number P04085) and B (PDGF-B; human PDGF-B has the UniProtKB/Swiss-Prot number P01127). Thereby, three protein dimers can be formed: PDGF-AA, PDGF-AB and PDGF-BB. Recently, two additional PDGF polypeptide chains, PDGF-C and PDGF-D, were identified. The currently known PDGF genes and polypeptides belong to a family of structurally and functionally related growth factors including also the vascular endothelial growth factors (VEGFs). PDGF/VEGF growth factors are conserved throughout the animal kingdom.
PDGFs act via two receptor tyrosine kinases (RTKs), PDGF receptor (PDGFR) alpha (PDGFRalpha) and beta (PDGFRbeta), with common domain structures, including five extracellular immunoglobulin (Ig) loops and a split intracellular tyrosine kinase domain. The VEGFs act through a distinct but structurally related subfamily of RTKs. Ligand binding promotes receptor dimerization, which initiates signaling. Depending on ligand configuration and the pattern of receptor expression, different receptor dimers may form. However, only a few interactions seem to be relevant in vivo; i.e., those of PDGF-AA and PDGF-CC via PDGFRalpha, and PDGF-BB via PDGFRbeta.
The PDGFs have crucial roles during development, but there is limited evidence for normal physiological functions in the adult. Studies of PDGFs and PDGFRs in animal development have revealed roles for PDGFRalpha signaling in gastrulation and in the development of the cranial and cardiac neural crest, gonads, lung, intestine, skin, CNS, and skeleton. Similarly, roles for PDGFRbeta signaling have been established in blood vessel formation and early hematopoiesis. PDGF signaling is implicated in a range of diseases. Autocrine activation of PDGF signaling pathways is involved in certain gliomas, sarcomas, and leukemias. Paracrine PDGF signaling is commonly observed in epithelial cancers, where it triggers stromal recruitment and may be involved in epithelial-mesenchymal transition, thereby affecting tumor growth, angiogenesis, invasion, and metastasis. PDGFs drive pathological mesenchymal responses in vascular disorders such as atherosclerosis, restenosis, pulmonary hypertension, and retinal diseases, as well as in fibrotic diseases, including pulmonary fibrosis, liver cirrhosis, scleroderma, glomerulosclerosis, and cardiac fibrosis.
Thus, increased PDGF activity has been linked with several diseases and pathological conditions. Causal pathogenic roles of the PDGFs have been established for some diseases, providing prospects for therapy using PDGF antagonists, such as PDGF specific antibodies. In addition, it has been suggested that the combination of anti-VEGF and anti-PDGF agents affords synergistic therapeutic benefits for treating certain ocular neovascular diseases (WO 2005/020972; Jo, N., Mailhos, C., Ju, M., Cheung, E., Bradley, J., Nishijima, K., Robinson, G. S., Adamis, A. P. and Shima, D. T., Am. J. Pathol., 168(6), 2036-2053, 2006).
There are, beside antibodies, novel binding proteins or binding domains that can be used to specifically bind a target molecule (e.g. Binz, H. K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005) and thereby act as an antagonist. One such novel class of binding proteins or binding domains not possessing an Fc are based on designed repeat proteins or designed repeat domains (WO 2002/020565; Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, M. T., Binz, H. K and Amstutz, P., Drug Discov. Today 13, 695-701, 2008). WO 2002/020565 describes how large libraries of repeat proteins can be constructed and their general application. Nevertheless, WO 2002/020565 does neither disclose the selection of repeat domains with binding specificity for PDGF-BB nor concrete repeat modules or repeat sequence motifs of repeat domains that specifically bind to PDGF-BB. Furthermore, WO 2002/020565 does not suggest that repeat domains with binding specificity for PDGF-BB could be used to regulate the PDGF-BB mediated signaling pathways to successfully treat diseases. These designed repeat domains harness the modular nature of repeat proteins and may possess N-terminal and C-terminal capping modules to prevent the designed repeat domains from aggregation by shielding the hydrophobic core of the domain (Forrer, P., Stumpp, M. T., Binz, H. K. and Plückthun, A., FEBS letters 539, 2-6, 2003).
The technical problem underlying the present invention is identifying novel binding proteins, such as ankyrin repeat proteins or domains, with binding specificity to PDGF-BB to regulate PDGF-BB mediated signaling pathways for an improved treatment of certain cancers, vascular disorders such as retinal diseases, fibrotic diseases and other pathological conditions. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.